Dokument: Untersuchung des Einflusses der Matrix und photoprotektiver Additive auf die Eigenschaften von Fluorophoren mittels Einzelmolekülspektroskopie
| Titel: | Untersuchung des Einflusses der Matrix und photoprotektiver Additive auf die Eigenschaften von Fluorophoren mittels Einzelmolekülspektroskopie | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=29495 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20140515-101425-8 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Deutsch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Doerr, Denis [Autor] | |||||||
| Dateien: |
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| Stichwörter: | Fluoreszenz, Fluoreszenzspektroskopie, Fluoreszenzkorrelationsspektroskopie, Einzelmolekülfluoreszenzspektroskopie, Photochemie, Fluoreszenzsignaloptiemierung, Fluorophore, Fluoreszenzsignal steigernde Additive, Mikrofluidik, Rheologie | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 540 Chemie | |||||||
| Beschreibungen: | Due to its easy detectability fluorescence is widely used in spectroscopy to investigate a variety of chemical and biochemical samples. The characteristics of fluorescence like intensity, lifetime, anisotropy and quantum yield contain information about electronic structure, mobility and orientation of fluorophores and they depend on the fluorophores environment. This interaction was used to characterize the environment as well as to tune the fluorophores.
The first goal of this work was to investigate the feasibility of fluorescence-based local force probes for polymers under tension, using the fluorescence signal of an oligoparaphenylenevinylene derivative (OPV 5) with a twisted molecular backbone. Improved optical sensors of this type should in principle be able to monitor local mechanical stress in transparent samples down to the single molecule level, which harbors promising applications in polymer and nano science and technology. To investigate correlations between the impact of an external mechanical force on the molecular framework of fluorophores and the resultant changes of their fluorescence properties OPV 5 was used to dope thin foils made of PVC. The foils were stretched by applying uniaxial force and three major optical effects were observed simultaneously. First, the fluorescence anisotropy increased, which indicates a reorientation of the fluorophores within the matrix. Second, the fluorescence lifetime decreased by approximately 2.5 % (25 ps). Finally, an increase of the emission energy of about 0.2% (corresponding to a blue-shift of 1.2 nm) was observed. In addition, analogous measurements with Rhodamine 123 as an inert reference dye showed only minor effects which can be attributed to matrix effects due to refractive index changes. To relate the observed spectroscopic changes to the underlying changes in molecular properties, the measured optical effects where compared to quantum-chemical calculations. Both, the observed blue-shift of fluorescence and the reduced lifetime of OPV 5 under tensile stress are consistent with the results of the semiempirical calculations. The accuracy of a fluorescence signal is limited by the number of detected photons, hence it is important to investigate and extend the fluorescence photon emission capabilities of fluorophores. Therefore the second goal of this work was to optimize the fluorescence signal of a fluorophore. In this context, three different additives which enhance the fluorescence signal were investigated as selective quenchers for triplet or radical states of Rhodamine 110 (Rh 110): 1) mainly triplet state quencher 4-(phenylazo)-benzoic acid (AZB-C) 2) the triplet and radical cation state quencher 4-(phenylazo)anilin-benzoic acid (A-AZB-C) 3) the strong antioxidant 6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid (Trolox). Fluorescence correlation spectroscopy (FCS) in combination with power plot analysis was used to describe the entire fluorescence output according to a derived kinetics model for excitation and fluorescence of Rh 110. Under air saturated conditions the triplet and radical cation states were observed as dark states of Rh 110, where the radical cation state was the major populated state. Hence the application of quencher A-AZB-C which effectively prevented the triplet and radical formation even at high excitation irradiance led to strongest enhancement of the fluorescence signal. At an excitation irradiance of 1 MW/cm² and 600 µM A-AZB-C a tenfold increase of the fluorescence countrate to approximately 2.4 MHz was observed. The application of the mainly triplet quenching AZB-C led to a significantly smaller enhancement of the fluorescence signal. At the same excitation irradiance of 1 MW/cm² and a concentration of 800 µM only a six times increased fluorescence countrate was observed. In case of application of the strong antioxidant Trolox the formation of an additional dark state, the radical anion state, was observed. Hence the enhancement of fluorescence signal with Trolox alone was similar to AZB-C. It was possible to quench effectively the radical anion state observed in the presence of Trolox with AZB-C. Consequently the enhancement of fluorescence signal in the presence of a mixture between Trolox and AZB-C was similar like with A-AZB-C alone. Additionally the photo destruction reaction of Rhodamine 123 (Rh 123) and the impact of the additives A-AZB-C and AZB-C on the photo stability of Rh 123 were investigated. It was found that the main pathway for the photo destruction reaction of Rh 123 in the presence of oxygen proceeds via the first excited singlet state of Rh 123. For this reason the investigated additives had only weak influence on the photo stability of Rh 123 in the presence of oxygen. Under deoxygenated conditions the photo stability of Rh 123 was increased by a factor of ten if the additive A-AZB-C or AZB-C with concentrations of 100 µM were added to the dye solution. Especially in single molecule detection (SMD) techniques with short dwell times of the fluorophores in the confocal volume, where only small numbers of fluorescence photons can be collected from each fluorophore, the enhancement of the fluorescence signal can significantly improve the measurements. Short dwell times can be also a result of directed flow. Therefore the third goal of this work was the application of additives for compensation of fluorescence photon loss under flow conditions. In this context, the fluorescence signals from Rh 123 and Rh 110 at flow conditions were investigated, using a combination of a single molecules detection fluorescence setup with a microfabricated diffusive mixer (micromixer). The quantitative analysis of SMD measurements in the micromixer requires the characterization of the flowpattern. One approach for calculating the flow in microchannels is Computational Fluid Dynamics (CFD). Here the fabrication precision of microchannels in general limits the accuracy of numerical calculations. At the same time FCS as experimental approach can probe the flow velocity in microchannels directly. However the characterization of flow pattern by FCS is limited by photon statistics and positioning accuracy of the confocal volume in the microchannels. At the same time the characterization of the flow pattern by FCS demands many single FCS measurements. Using the complementary combination of CFD with FCS it was possible to overcome most of the limitations of both methods. Thus a small number of FCS measurements provided necessary information for accurate numerical calculation of the flow pattern. Vice versa the knowledge of the flow pattern in combination with FCS provided the accurate position of the confocal volume inside of a microchannel. Moreover using the calculated flow velocity for calibration of the confocal volume in single focus FCS measurements it was possible to estimate the precise value for the diffusion coefficient of Rh 110 The time resolution of the micromixer is set by the time required for diffusively mixing and the dwell time needed to collect a given number of photons. In fact, the flow induced photon loss limited the maximum flow velocity for SMD measurements to 4 µm/ms, which degraded the maximum time resolution of the used micromixer from 1.8 ms to 3 ms. One approach to collect enough fluorescence photons at high flow is to increase the excitation intensity, however this alone is unsatisfactory since it increases the probability to populate long lived triplet and radical states. The application of the dark state quencher A-AZB-C with 400 µM concentration in combination with a high excitation irradiance of 170 kW/cm² SMD measurements of Rh 123 improved the fluorescence signal by a factor of four. Thereby the flow induced photon loss was compensated, so that the time resolution of the micromixer was finally limited only by the time required for diffusively mixing.Aufgrund ihrer einfachen Detektierbarkeit und hohen Empfindlichkeit spielt die Fluoreszenz eine wichtige Rolle bei der Untersuchung chemischer und biologischer Proben. Die spektralen Eigenschaften der Fluoreszenz, die Fluoreszenzlebensdauer, die Fluoreszenzanisotropie und die Fluoreszenzquantenausbeute enthalten wertvolle Informationen über die elektronische Struktur, die Beweglichkeit und die räumliche Orientierung der Fluorophore. Daher wird die Fluoreszenz im Rahmen dieser Arbeit eingesetzt, um Informationen über Fluorophore und ihre Umgebung zu gewinnen. Zuerst wurde die Eignung eines Oligophenylenvinylenderivats (OPV 5) als Fluoreszenz-basiertem Kraftsensor in einer Polyvinylchlorid-Folie untersucht. Bei einer Verformung der mit OPV 5 dotierten Folie konnte eine kraftabhängige Verkürzung der Fluoreszenzlebensdauer und eine Blauverschiebung des Fluoreszenzemissionsspektrums gemessen werden. Beide Effekte konnten durch die entsprechenden semiempirischen quantenmechanischen Simulationen auf die Wirkung der mechanischen Kraft auf das verdrillte Molekülgerüst von OPV 5 zurückgeführt werden. Die Genauigkeit der aus der Fluoreszenz gewonnenen Informationen anhand der charakteristischen Parameter ist durch die Anzahl der detektierten Fluoreszenzphotonen limitiert. Daher wurde als Nächstes die fluoreszenzsignalsteigernde Wirkung von drei chemischen Additiven mit unterschiedlichen Redoxpotentialen 4-[(4-Aminophenyl)diazenyl]benzoesäure (A-AZB-C), 4-(Phenylazo)-benzoesäure (AZB-C) und 6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonsäure (Trolox) auf den Fluorophor Rhodamin 110 (Rh 110) untersucht. Dazu wurde ein umfassendes photochemisches Reaktionsschema entwickelt, um die Effekte der Additive auf Rh 110 quantitativ zu beschreiben. Schließlich konnte eine mehr als zehnfache Steigerung des Fluoreszenzsignals durch den Zusatz von A-AZB-C oder einer Trolox/AZB-C-Mischung erreicht werden. Als Letztes wurde das Fluoreszenzsignal des Rh 110 im Rahmen dieser Arbeit eingesetzt um Flussprofile in den Kanälen eines mikrofluidischen Mixers zu charakterisieren, wobei eine Kombination aus Fluoreszenzkorrelationsspektroskopie (FCS) und numerischen Simulationen verwendet wurde. Der Fluss verkürzt jedoch die Verweildauer der Fluorophore im Detektionsvolumen und verringert somit die detektierte Fluoreszenz¬photonenzahl bei Einzelmolekülfluoreszenzmessungen, was die kinetische Zeitauflösung des Mixers verschlechtert. In diesem Zusammenhang konnte das Additiv A-AZB-C bei Einzelmolekülmessungen von Rhodamin 123 erfolgreich unter Flussbedingungen eingesetzt werden, um den flussbedingten Fluoreszenzphotonenverlust zu kompensieren und so die Limitation der Zeitauflösung durch die Fluoreszenzdetektion auch bei hohen Flüssen zu überwinden. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Chemie » Physikalische Chemie und Elektrochemie | |||||||
| Dokument erstellt am: | 15.05.2014 | |||||||
| Dateien geändert am: | 15.05.2014 | |||||||
| Promotionsantrag am: | 04.11.2013 | |||||||
| Datum der Promotion: | 19.12.2013 |
